Pks21221

T cells were isolated from human PBMCs using Human T-cell Isolation kit (StemCell Technologies, Cat#17951). The purity of T cells was greater than 90% as confirmed by flow cytometry. Cells were counted and loaded with 5 μM CellTrace Violet (Thermo Fisher) in phosphate buffered saline (PBS) for 10 min at 37 °C, followed by wash in 10% fetal calf serum (FCS)-supplemented RPMI 1640 media. T cells were cultured in 10% HS-RPMI, with anti-CD3 (1 μg per mL, eBioscience) and anti-CD28 (1 μg per mL, eBioscience), in the presence or absence of different concentration of PKS21221 or Bortezomib (Selleckchem). In some experiments, T cells were cultured in 10% HS-RPMI / anti-CD3 / anti-CD28 for 4 days and then treated with PKS21221 or Bortezomib (Selleckchem) for 12, 24, 48 h. The viability and apoptosis were assessed using Annexin V Apoptosis Detection Kit (BD Biosciences). The experiments were performed in cells from three different healthy volunteer donors.

Human PBMCs were obtained from 5 different healthy volunteers as above. Isolated PBMCs were incubated in 10% HS-RPMI at 5 × 10⁵ cells per 200 μl per well in 96-well round bottom plate (Corning), for 12, 24, 48 h, in the presence of different concentrations of PKS21221 or Bortezomib (Selleckchem). The viability of different populations of PBMCs, such as T cells (CD3⁺), B cells (CD19⁺CD3⁻CD14⁻), monocytes (CD14⁺), dendritic cells (CD3⁻CD19⁻CD14⁻CD16⁻HLADR⁺CD11c⁺), were assessed by flow cytometry using Fixable Viability dye (Themo Fisher).